Cyclic decapeptide antibiotics

ABSTRACT

Several cyclic decapeptides having antibiotic activity are disclosed. The decapeptides are active against both gram positive and gram negative bacteria.

TECHNICAL FIELD

The present invention relates to novel cyclic peptide compounds withpotent activity against antibiotic-resistant pathogens.

BACKGROUND OF THE INVENTION

Methicillin-resistant strains of Staphylococcus aureus (USA) causeinfections that are refractory to standard anti-staphylococciantibiotics, and in many cases vancomycin is the antibiotic of lastresort. Consequently, it is of great concern that vancomycin-resistantstrains of MRSA may develop.

Infections due to enterococci have been difficult to treat for manyyears because these organisms are intrinsically resistant to manyantibiotics. Ampicillin has been the mainstay for treatment ofuncomplicated enterococcal infections, but many strains have now becomeresistant to ampicillin. Vancomycin is again the only effectivetreatment for these ampicillin-resistant enterococcal infections. In thepast few years, vancomycin-resistant enterococcal strains (VRE) havebegun to appear and they are rapidly spreading across North America.There are no effective antibiotics currently available for suchorganisms and the recent report of an outbreak of VRE with a 73%mortality rate has highlighted the seriousness of the situation. SeeEdmond, M. B. et al., Clinical Infectious Diseases 20:1126-33, 1995.

One area where new drugs are desperately needed is in the treatment ofantibiotic-resistant strains of gram positive human pathogens. Thepresent invention is directed to fulfilling this need, and providesrelated advantages as described herein.

SUMMARY OF THE INVENTION

In a first aspect, the invention is directed to isolated cyclicdecapeptides of the formulas (1), (2) and (3):

(1): cyclo [L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-Asp-L-Tyr](SEQ ID NO:1);

(2): cyclo [L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-Trp](SEQ ID NO:2); and

(3) cyclo [L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Trp-D-Phe-L-Asn-L-Asp-L-Trp](SEQ ID NO:3).

The decapeptide of Formula (1) is referred to herein as Loloatin A. Thedecapeptide of Formula (2) is referred to herein as Loloatin B. Thedecapeptide of Formula (3) is referred to herein as Loloatin C.

The invention is directed to the above-identified cyclic decapeptides inan isolated, i.e., substantially purified form, preferably in a quantityof more than about 1 gram, more preferably in a quantity of more thanabout 10 grams, still more preferably in a quantity of more than about100 grams, and yet still more preferably in a quantity of more thanabout 1 kilogram. A substantially purified form is a composition whereinthe above-listed cyclic decapeptides constitute at least about 1 weightpercent of the composition, preferably at least about 10 weight percent,more preferably at least about 30 weight percent, still more preferablyat least about 50 weight percent, yet still more preferably at leastabout 70 weight percent, and yet still more preferably at least about 95weight percent, and most preferably at least about 99 weight percent.

The invention is also directed to the above-identified cyclicdecapeptides in a pharmaceutical composition. A pharmaceuticalcomposition of the invention may not necessarily contain the cyclicdecapeptide in a substantially purified form because the composition maycontain diluent and/or other materials commonly found in pharmaceuticalcompositions.

The invention is also directed to a method of treating bacterialinfection, comprising administering to a patient having a bacterialinfection, an amount of the above-identified cyclic decapeptideseffective to relieve symptoms associated with or due to the bacterialinfection.

In another aspect, the invention is directed to various derivatives andanalogs of Loloatin A, B and C.

Loloatin derivatives of the invention include the solvates, salts(either acid- or base- addition salts, depending on whether the aminoacid sidechain being converted to a salt is basic or acidic,respectively), esters (derivatives of amino acid sidechains containing acarboxylic acid group), amines (derivatives of amino acid sidechainscontaining an amino group), ethers (derivatives of amino acid sidechainscontaining an hydroxyl group) and amides (derivatives of amino acidsidechains containing either an amine or carboxylic acid group) ofLoloatin A, B or C.

For example, the invention provides for a salt of a compound selectedfrom the group:

cyclo[L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-Tyr] (SEQID NO:1);

cyclo[L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-Trp] (SEQID NO:2); and

cyclo[L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Trp-D-Phe-L-Asn-L-Asp-L-Trp] (SEQID NO:3).

The salt may contain at least one negatively charged ion selected fromchloride, bromide, sulfate, phosphate, C₁₋₁₅carboxylate,methanesulfonate and p-toluenesulfonate, which are exemplary only.Exemplary C₁₋₁₅carboxylates include acetate, glycolate, lactate,pyruvate, malonate, succinate, glutarate, fumarate, malate, tartarate,citrate, ascorbate, maleate, hydroxymaleate, benzoate, hydroxybenzoate,phenylacetate, cinnamate, salicylate and 2-phenoxybenzoate.

The salt may contain at least one positively charged ion selected fromlithium, sodium, potassium, beryllium, magnesium, calcium and quaternaryammonium ions, which are exemplary positively charged ions. Exemplaryquaternary ammonium ions cinlude tetraallylammonium, andtrialkylaralkylammonium ions.

The invention also provides for derivatives of a compound selected from

cyclo[L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-Tyr] (SEQID NO:1);

cyclo[L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-Trp] (SEQID NO:2); and

cyclo[L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Trp-D-Phe-L-Asn-L-Asp-L-Trp] (SEQID NO:3).

For example, the derivative may have the amine group of the ornithinesidechain being a secondary, tertiary or quaternary amine group, andother amino acid sidechains are optionally in a derivative form as well.Exemplary ornithine sidechains have the formula —CH₂—CH₂—CH₂—NHR³,—CH₂—CH₂—CH₂—N(R³)₂ or —CH₂—CH₂—CH₂—N(R³)₃ wherein R³ is an alkyl groupthat may be of straight chain, or where possible, of cyclic or branchedstructure and may contain one or more alkene, alkyne, or aromaticfunctionalities; or an acyl group that may be of straight chain, orwhere possible, of cyclic or branched structure and may contain one ormore alkene, alkyne, or aromatic functionalities.

In other derivatives, the hydroxyl group of one or more tyrosinesidechains has been converted to an ether or ester group, and otheramino acid sidechains are optionally in a derivative form as well. Forexample, at least one tyrosine sidechain may have the formula—CH₂—C₆H₄—O—R², wherein C₆H₄ is an aromatic ring and —O—R² is in thepara position, and R² is a C₁₋₁₅ alkyl group so as to form an ether,where the alkyl group may be of straight chain, or where possible, ofcyclic or branched structure and may contain one or more alkene, alkyne,or aromatic functionalities; or a C₁₋₁₅ acyl group so as to form anester, where the acyl group may be of straight chain, or where possible,of cyclic or branched structure and may contain one or more alkene,alkyne, or aromatic functionalities.

In other derivatives, the carboxyl group of the aspartic acid sidechainhas been converted to an amide or ester group, and other amino acidsidechains are optionally in a derivative form as well. For example, theaspartic acid sidechain may have the formula —CH₂—C(═O)O—R¹, and R¹ is aC₁₋₁₅ alkyl group that may be of straight chain, or where possible, ofcyclic or branched structure and may contain one or more alkene, alkyne,or aromatic functionalities; or an aryl group. In another example, theaspartic acid sidechain has the formula —CH₂—C(═O)N—R¹, and R¹ is aC₁₋₁₅ alkyl group that may be of straight chain, or where possible, ofcyclic or branched structure and may contain one or more alkene, alkyne,or aromatic functionalities; or an aryl group.

The invention also provides for a pharmaceutical composition comprisinga salt, derivative or analog of any of Loloatin A, Loloatin B andLoloatin C, as set forth above, in admixture with a pharmaceuticallyacceptable carrier.

The invention also provides for a method for the treatment of a patientafflicted with a bacterial infection comprising the administration tosaid patient of a therapeutically effective amount of a salt, derivativeor analog of any of Loloatin A, Loloatin B and Loloatin C, as set forthabove

The invention also provides for the use of a salt, derivative or analogof any of Loloatin A, Loloatin B and Loloatin C, as set forth above, inthe manufacture of a medicament, where the medicament may be used totreat bacterial infection.

The Loloatin analogs include cyclic decapeptides having a “non-natural”stereochemistry at one or more of the α-carbons of the component aminoacids, where the “natural” stereochemistry is as indicated by the L- orD- designations preceding the name of each of the amino acids in theformulas for Loloatin A, B and C set forth above. Collectively, theseLoloatin analogs are represented by the formulae:

cyclo[Val-Orn-Leu-Tyr-Pro-Phe-Phe-Asn-Asp-Try] (SEQ ID NO:75);

cyclo[Val-Orn-Leu-Tyr-Pro-Phe-Phe-Asn-Asp-Trp] (SEQ ID NO:76); and

cyclo[Val-Orn-Leu-Tyr-Pro-Trp-Phe-Asn-Asp-Trp] (SEQ ID NO:77).

Furthermore, the invention is directed to various analogs of theabove-identified compounds, where preferred analogs have the formulaslisted below. In the below-listed structures, no stereochemistry isdesignated because the analogs of the invention may have any possiblestereochemistry at each atom capable of having more than onestereochemical arrangement of substituents. However, looking at thebelow listed sequences from left to right as written, preferred analogshave the stereochemistry L-, L-, L-, D-, L-, L-, D-, L-, L- and L-. Forexample, cyclo[Val-Orn-Leu-Tyr-Pro-Trp-Phe-Asn-Asp-β-Phenylserine] (SEQID NO:74), as written below preferably has the stereochemistrycyclo[L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Trp-D-Phe-L-Asn-L-Asp-L-β-Phenylserine](SEQ ID NO:74).

Preferred analogs have one amino acid residue present in Loloatin A, Bor C replaced with a different amino acid residue. Preferred analogsare:

cyclo[Butyrine-Orn-Leu-Tyr-Pro-Phe-Phe-Asn-Asp-Tyr] (SEQ ID NO:4),

cyclo[Butyrine-Orn-Leu-Tyr-Pro-Phe-Phe-Asn-Asp-Trp] (SEQ ID NO:5);

cyclo[Butyrine-Orn-Leu-Tyr-Pro-Trp-Phe-Asn-Asp-Trp] (SEQ ID NO:6);

cyclo[Val-diaminobutyric aicd-Leu-Tyr-Pro-Phe-Phe-Asn-Asp-Tyr] (SEQ IDNO:7);

cyclo[Val diaminobutyric aicd-Leu-Try-Pro-Phe-Phe-Asn-Asp-Trp] (SEQ IDNO:8);

cyclo[Val-diaminobutyric aicd-Leu-Tyr-Pro-Trp-Phe-Asn-Asp-Trp] (SEQ IDNO:9);

cyclo[Val-Orn-Isoleucine-Tyr-Pro-Phe-Phe-Asn-Asp-Tyr] (SEQ ID NO:10);

cyclo[Val-Orn-Isoleucine-Tyr-Pro-Phe-Phe-Asn-Asp-Trp] (SEQ ID NO:11);

cyclo[Val-Orn-Isoleucine-Tyr-Pro-Trp-Phe-Asn-Asp-Trp] (SEQ ID NO:12);

cyclo[Val-Orn-Alloisoleucine-Tyr-Pro-Phe-Phe-Asn-Asp-Tyr] (SEQ IDNO:13);

cyclo[Val-Orn-Alloisoleucine-Tyr-Pro-Phe-Phe-Asn-Asp-Tyr] (SEQ IDNO:14);

cyclo[Val-Orn-Alloisoleucine-Tyr-Pro-Trp-Phe-Asn-Asp-Trp] (SEQ IDNO:15);

cyclo[Val-Orn-Norvaline-Tyr-Pro-Phe-Phe-Asn-Asp-Tyr] (SEQ ID NO:16);

cyclo[Val-Orn-Norvaline-Tyr-Pro-Phe-Phe-Asn-Asp-Trp] (SEQ ID NO:17);

cyclo[Val-Orn-Norvaline-Tyr-Pro-Trp-Phe-Asn-Asp-Trp] (SEQ ID NO:18);

cyclo[Val-Orn-Cyclopropylalanine-Tyr-Pro-Phe-Phe-Asn-Asp-Tyr] (SEQ IDNO:19);

cyclo[Val-Orn-Cyclopropylalanine-Tyr-Pro-Phe-Phe-Asn-Asp-Trp] (SEQ IDNO:20);

cyclo[Val-Orn-Cyclopropylalanine-Tyr-Pro-Trp-Phe-Asn-Asp-Trp] (SEQ IDNO:21);

cyclo[Val-Orn-Norleucine-Tyr-Pro-Phe-Phe-Asn-Asp-Tyr] (SEQ ID NO:22);

cyclo[Val-Orn-Norleucine-Try-Pro-Phe-Phe-Asn-Asp-Trp] (SEQ ID NO:23);

cyclo[Val-Orn-Norleucine-Tyr-Pro-Trp-Phe-Asn-Asp-Trp] (SEQ ID NO:24);

cyclo[Val-Orn-Norleucine-Tyr-Pro-Phe-Phe-Asn-Asp-Tyr] (SEQ ID NO:22);

cyclo[Val-Orn-Norleucine-Tyr-Pro-Phe-Phe-Asn-Asp-Trp] (SEQ ID NO:23);

cyclo[Val-Orn-Norleucine-Tyr-Pro-Trp-Phe-Asn-Asp-Trp] (SEQ ID NO:24);

cyclo[Val-Orn-Leu-p-fluorophenylalamine-Pro-Phe-Phe-Asn-Asp-Tyr] (SEQ IDNO:25);

cyclo[Val-Orn-Leu-p-fluorophenylalamine-Pro-Phe-Phe-Asn-Asp-Trp] (SEQ IDNO:26);

cyclo[Val-Orn-Leu-p-fluorophenylalamine-Pro-Trp-Phe-Asn-Asp-Trp] (SEQ IDNO:27);

cyclo[Val-Orn-Leu-Tryptophan-Pro-Phe-Phe-Asn-Asp-Tyr] (SEQ ID NO:28);

cyclo[Val-Orn-Leu-Tryptophan-Pro-Phe-Phe-Asn-Asp-Trp] (SEQ ID NO:29);

cyclo[Val-Orn-Leu-Tryptophan-Pro-Trp-Phe-Asn-Asp-Trp] (SEQ ID NO:30);

cyclo[Val-Orn-Leu-Thienylalanine-Pro-Phe-Phe-Asn-Asp-Tyr] (SEQ IDNO:31);

cyclo[Val-Orn-Leu-Thienylalanine-Pro-Phe-Phe-Asn-Asp-Trp] (SEQ IDNO:32);

cyclo[Val-Orn-Leu-Thienylalanine-Pro-Trp-Phe-Asn-Asp-Trp] (SEQ IDNO:33);

cyclo[Val-Orn-Leu-Tyr-Azetidine-2-carboxylic acid-Phe-Phe-Asn-Asp-Tyr](SEQ ID NO:34);

cyclo[Val-Orn-Leu-Tyr-Azetidine-2-carboxylic acid-Phe-Phe-Asn-Asp-Trp](SEQ ID NO:35);

cyclo[Val-Orn-Leu-Tyr-Azetidine-2-carboxylic acid-Trp-Phe-Asn-Asp-Trp](SEQ ID NO:36);

cyclo[Val-Orn-Leu-Tyr-Pipecolic acid-Phe-Phe-Asn-Asp-Tyr] (SEQ IDNO:37);

cyclo[Val-Orn-Leu-Tyr-Pipecolic acid-Phe-Phe-Asn-Asp-Trp] (SEQ IDNO:38);

cyclo[Val-Orn-Leu-Tyr-Pipecolic acid-Trp-Phe-Asn-Asp-Trp] (SEQ IDNO:39);

cyclo[Val-Orn-Leu-Tyr-trans-3-Methylproline-Phe-Phe-Asn-Asp-Tyr] (SEQ IDNO:40);

cyclo[Val-Orn-Leu-Tyr-trans-3-Methylproline-Phe-Phe-Asn-Asp-Trp] (SEQ IDNO:41);

cyclo[Val-Orn-Leu-Tyr-trans-3-Methylproline-Trp-Phe-Asn-Asp-Trp] (SEQ IDNO:42);

cyclo[Val-Orn-Leu-Tyr-trans-4-Fluoroproline-Phe-Phe-Asn-Asp-Tyr] (SEQ IDNO:43);

cyclo[Val-Orn-Leu-Tyr-trans-4-Fluoroproline-Phe-Phe-Asn-Asp-Trp] (SEQ IDNO:44);

cyclo[Val-Orn-Leu-Tyr-trans-4-Fluoroproline-Trp-Phe-Asn-Asp-Trp] (SEQ IDNO:45);

cyclo[Val-Orn-Leu-Tyr-Pro-Tyr-Phe-Asn-Asp-Tyr] (SEQ ID NO:46);

cyclo[Val-Orn-Leu-Tyr-Pro-Tyr-Pher-Asn-Asp-Trp] (SEQ ID NO:47);

cyclo[Val-Orn-Leu-Tyr-Pro-p-Fluorophenylalanine-Phe-Asn-Asp-Tyr] (SEQ IDNO:48);

cyclo[Val-Orn-Leu-Tyr-Pro-p-Fluorophenylalanine-Phe-Asn-Asp-Trp] (SEQ IDNO:49);

cyclo[Val-Orn-Leu-Tyr-Pro-Trp-Phe-Asn-Asp-Tyr] (SEQ ID NO:50);

cyclo[Val-Orn-Leu-Tyr-Pro-Trp-Phe-Asn-Asp-Trp] (SEQ ID NO:51);

cyclo[Val-Orn-Leu-Tyr-Pro-Thienylalanine-Phe-Asn-Asp-Tyr] (SEQ ID NO:52)

cyclo[Val-Orn-Leu-Tyr-Pro-Thienylalanine-Phe-Asn-Asp-Trp] (SEQ ID NO:53)

cyclo[Val-Orn-Leu-Tyr-Pro-β-Phenylserine-Phe-Asn-Asp-Tyr] (SEQ IDNO:54);

cyclo[Val-Orn-Leu-Tyr-Pro-β-Phenylserine-Phe-Asn-Asp-Trp] (SEQ IDNO:55);

cyclo[Val-Orn-Leu-Tyr-Pro-Phe-Tyr-Asn-Asp-Tyr] (SEQ ID NO:56);

cyclo[Val-Orn-Leu-Tyr-Pro-Phe-Tyr-Asn-Asp-Trp] (SEQ ID NO:57);

cyclo[Val-Orn-Leu-Try-Phe-p-Fluorophenylalanine-Asn-Asp-Tyr] (SEQ IDNO:58);

cyclo[Val-Orn-Leu-Try-Phe-p-Fluorophenylalanine-Asn-Asp-Trp] (SEQ IDNO:59);

cyclo[Val-Orn-Leu-Tyr-Pro-Phe-Trp-Asn-Asp-Tyr] (SEQ ID NO:60);

cyclo[Val-Orn-Leu-Tyr-Pro-Phe-Trp-Asn-Asp-Trp] (SEQ ID NO:61);

cyclo[Val-Orn-Leu-Tyr-Pro-Phe-Thienylalanine-Asn-Asp-Tyr] (SEQ IDNO:62);

cyclo[Val-Orn-Leu-Tyr-Pro-Phe-Thienylalanine-Asn-Asp-Trp] (SEQ IDNO:63);

cyclo[Val-Orn-Leu-Try-Pro-Phe-β-Phenylserine-Asn-Asp-Tyr] (SEQ IDNO:64);

cyclo[Val-Orn-Leu-Try-Pro-Phe-β-Phenylserine-Asn-Asp-Trp] (SEQ IDNO:65);

cyclo[Val-Orn-Leu-Tyr-Pro-Trp-Phe-Asn-Asp-Tyr] (SEQ ID NO:66);

cyclo[Val-Orn-Leu-Tyr-Pro-Phe-Phe-Asn-Asp-p-Fluorophenylalanine] (SEQ IDNO:67);

cyclo[Val-Orn-Leu-Tyr-Pro-Trp-Phe-Asn-Asp-p-Fluorophenylalanine] (SEQ IDNO:68);

cyclo[Val-Orn-Leu-Tyr-Pro-Phe-Phe Asn-Asp-Phe] (SEQ ID NO:69);

cyclo[Val-Orn-Leu-Tyr-Pro-Trp-Phe-Asn-Asp-Phe] (SEQ ID NO:70);

cyclo[Val-Orn-Leu-Tyr-Pro-Phe-Phe-Asn-Asp-Thienylalanine] (SEQ IDNO:71);

cyclo[Val-Orn-Leu-Tyr-Pro-Trp-Phe-Asn-Asp-Thienylalanine] (SEQ IDNO:72);

cyclo[Val-Orn-Leu-Tyr-Pro-Phe-Phe-Asn-Asp-β-Phenylserine] (SEQ IDNO:73); and

cyclo[Val-Orn-Leu-Tyr-Pro-Trp-Phe-Asn-Asp-β-Phenylserine] (SEQ IDNO:73).

The present invention is also directed to derivatives of theabove-listed Loloatin analogs (i.e., analog derivatives), including thesolvates, salts (either acid- or base- addition salts, depending onwhether the amino acid sidechain being converted to a salt is basic oracidic, respectively), esters (derivatives of amino acid sidechainscontaining a carboxylic acid group), amine (derivatives of amino acidsidechains containing an amino group), ethers (derivatives of amino acidsidechains containing an hydroxyl group) and amides (derivatives ofamino acid sidechains containing either an amine or carboxylic acidgroup) of the Loloatin A, B and C analogs listed above.

Collectively, the above-identified Loloatin A, B and C derivatives,analogs and analog derivatives are referred to herein as compounds ofFormula (A), or compounds of the invention.

The invention is also directed to the compounds of the invention in anisolated, i.e., substantially purified form, preferably in a quantity ofmore than about 1 gram, more preferably in a quantity of more than about10 grams, still more preferably in a quantity of more than about 100grams, and yet still more preferably in a quantity of more than about 1kilogram. A substantially purified form is a composition wherein theabove-listed compound of the invention constitutes at least about 1weight percent of the composition, preferably at least about 10 weightpercent, more preferably at least about 30 weight percent, still morepreferably at least about 50 weight percent, yet still more preferablyat least about 70 weight percent, and yet still more preferably at leastabout 95 weight percent, and most preferably at least about 99 weightpercent.

The invention is also directed to pharmaceutical compositions comprisingcompounds of the invention.

The invention is also directed to a method of treating bacterialinfection, comprising administering to a patient having a bacterialinfection, an amount of a compound of the invention effective to relievesymptoms associated with or due to the bacterial infection.

The compounds of the present invention contain multiple asymmetriccarbon atoms and thus exist as enantiomers and diastereomers. Unlessotherwise noted, the present invention includes all enantiomeric anddiastereomeric forms of the compounds. Pure stereoisomers, mixtures ofenantiomers and/or diastereomers, and mixtures of different compounds ofthe invention are included within the scope of the present invention.

The synthesis procedures described herein, especially when taken withthe general knowledge in the art, provide sufficient guidance to thoseof ordinary skill in the art to perform the synthesis, isolation, andpurification of the compounds described herein and other analogouscompounds. Individual enantiomers may be obtained, if desired, frommixtures of the different forms by known methods of resolution, such asthe formation of diastereomers, followed by recrystalisation.Alternatively, isomerically pure starting materials may be employed inthe synthesis of a compound of the invention.

The compounds of the invention may be in the form of a solvate or apharmaceutically acceptable salt, e.g., an acid- or base- addition salt.Such salts may have at least one negatively charged ion such aschloride, bromide, sulfate, phosphate, C₁₋₁₅carboxylate,methanesulfonate and p-toluenesulfonate, where exemplaryC₁₋₁₅carboxylate ions are acetate, glycolate, lactate, pyruvate,malonate, succinate, glutarate, fumarate, malate, tartarate, citrate,ascorbate, maleate, hydroxymaleate, benzoate, hydroxybenzoate,phenylacetate, cinnamate, salicylate and 2-phenoxybenzoate. The salt mayhave at least one positively charged ion such as lithium, sodium,potassium, beryllium, magnesium, calcium and quaternary ammonium ions,where exemplary quaternary ammonium ions are tetraalkylammonium, andtrialkylaralkylammonium ions.

The invention is also directed to the above-identified cyclicdecapeptides and derivatives thereof in a pharmaceutical composition. Apharmaceutical composition of the invention may not necessarily containthe cyclic decapeptide or derivative thereof in a substantially purifiedform because the composition may contain diluent and/or other materialscommonly found in pharmaceutical compositions, such that theabove-identified cyclic decapeptides and derivatives thereof constituteless that 1 weight percent of the pharmaceutical composition.

The invention is also directed to a method of employing the compounds ofthe invention as an antibiotic.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to isolated cyclic decapeptides having one offormulas (1), (2) or (3) shown below (SEQ ID NO:1, 2 and 3 respectively)and are named Loloatin A, Loloatin B and Loloatin C, respectively.

Written in different terms, Loloatin A, B and C have the amino acidsequences set forth in Table A below, where the first listed amino acidis joined through a peptide bond to the last listed amino acid so as toform a cyclic structure.

TABLE A Loloatin A Loloatin B Loloatin C L-Val L-Val L-Val L-Orn L-OrnL-Orn L-Leu L-Leu L-Leu D-Tyr D-Tyr L-Tyr L-Pro L-Pro L-Pro L-Phe L-PheL-Trp D-Phe D-Phe D-Phe L-Asn L-Asn L-Asn L-Asp L-Asp L-Asp L-Tyr L-TrpL-Trp

In another aspect, the invention is directed to cyclic compoundsrepresented by the shorthand structure shown below, denoted Formula (A).

In Formula (A), AA¹ through AA¹⁰ are generic symbols, each representingan amino acid residue as defined herein, or a salt or derivativethereof. Each line between neighboring (attached) AA¹-AA¹⁰ residuesrepresents an amide (also known as a peptide) bond formed betweenneighboring AA¹-AA¹⁰ residues, as well as the isosteres thereof“Isostere” means a modified form of the normal peptide bond (—C(O)NH—)between attached amino acid residues, such as —CH₂NH— (reduced),C(O)N(CH₃) (N-methylamide), —COCH₂— (keto), —CH(OH)CH₂— (hydroxy),—CH(NH₂)CH₂— (amino), —CH₂CH₂— (hydrocarbon), or —NHC(O)— (invertednormal peptide bond). Preferably the compounds of the present inventionare not in isosteric forms.

In Formula (A), AA¹-AA¹⁰ represent residues from the following specificamino acids or other listed compounds, where stereochemical designationsare preferred only, and the specifically named amino acid or otherlisted compound may be in either the L or D form:

AA¹: L-valine, butyrine;

AA²: L-ornithine, L-diaminobutyric acid;

AA³: L-teucine, L-isoleucine, L-alloisoleucine, L-norvaline,L-cyclopropylalanine, norleucine;

AA⁴: D-tyrosine, p-fluorophenylalanine, tryptophan, thienylalanine;

AA⁵: L-proline, azetidine-2-carboxylic acid, pipecolic acid,trans-3-methylproline, trans-fluoroproline;

AA⁶: L-phenylalanine, tryptophan, tyrosine, p-fluorophenylalanine,thienylalanine, β-phenylserine;

AA⁷: D-phenylalanine, tyrosine, p-fluorophenylalanine, tryptophan,thienylalanine, β-phenylserine;

AA⁸: L-asparagine;

AA⁹: L-aspartic acid or esters thereof; and

AA¹⁰: L-tryptophan, L-tyrosine, p-fluorophenylalanine, phenylalanine,thienylalanine, β-phenylserine.

The compounds of Formula (A) specifically include salts and otherderivatives of the amino acids listed above. An amino acid derivative isintended to include the solvates, salts (either acid- or base- additionsalts, depending on whether the amino acid sidechain is basic or acidic,respectively), esters (derivatives of amino acid sidechains containing acarboxylic acid group), amines (derivatives of amino acid sidechainscontaining an amino group), ethers (derivatives of amino acid sidechainscontaining an hydroxyl group) and amides (derivatives of amino acidsidechains containing either an amine or carboxylic acid group) of theunmodified cyclic compound. Collectively, the compounds of the inventionmay be referred to as compounds of Formula (A).

Except where otherwise stated, throughout this specification therecitation of a compound denotes all possible isomers within thestructural formula given for those compounds, in particular opticalisomers. Unless otherwise stated definitions are to be regarded ascovering mixtures of isomers, and individual isomers, including racemicmixtures, where they can be resolved.

Except if otherwise stated, definitions of compounds in thisspecification are to be regarded as covering all possible esters of thecompounds. In particular, except if otherwise stated, the recitation ofan amino acid residue having a carboxylic acid group is to be regardedas a recitation of all possible esters of that carboxylic acid.

Except if otherwise stated, definitions of compounds in thisspecification having phenolic groups are to be regarded as covering allpossible ethers or esters of the phenolic hydroxyl group.

Preferred compound of the invention have one of formulas (4), (5) or (6)shown below (SEQ ID NOS:1,2 and 3 respectively:

wherein

R¹ represents a hydrogen atom; or an alkyl group that may be of straightchain, or where possible, of cyclic or branched structure and maycontain one or more alkene, alkyne, or aromatic functionalities; or anaryl group.

R² represents a hydrogen atom; or an alkyl group that may be of straightchain, or where possible, of cyclic or branched structure and maycontain one or more alkene, alkyne, or aromatic functionalities; or anacyl group that may be of straight chain, or where possible, of cyclicor branched structure and may contain one or more alkene, alkyne, oraromatic functionalities.

R³ represents a hydrogen atom; or an alkyl group that may be of straightchain, or where possible, of cyclic or branched structure and maycontain one or more alkene, alkyne, or aromatic functionalities; or anacyl group that may be of straight chain, or where possible, of cyclicor branched structure and may contain one or more alkene, alkyne, oraromatic functionalities.

The compounds of the invention may also be described in terms of Formula(B)

wherein:

R⁴ is C₁₋₅alkyl, which may be branched or linear, or C₃₋₅cycloalkyl;

R⁵ is —(CH₂)_(n)—N(R₁₅)₂ wherein n is 1, 2, 3 or 4 and R₁₅ isindependently hydrogen or C₁₋₁₅alkyl group that may be of straightchain, or where possible, of cyclic or branched structure and maycontain one or more alkene, alkyne, or aromatic functionalities; or aC₁₋₁₅acyl group that may be of straight chain, or where possible, ofcyclic or branched structure and may contain one or more alkene, alkyne,or aromatic functionalities;

R⁶ is C₁₋₇alkyl, which may be branched or linear, or C₃₋₇cycloalkyl;

R⁷ is CH₂)_(m)—C₆H₄—OR₁₆ wherein m is 1, 2 or 3 and R₁₆ is hydrogenatom; or a C₁₋₁₅ alkyl group that may be of straight chain, or wherepossible, of cyclic or branched structure and may contain one or morealkene, alkyne, or aromatic functionalities; or a C₁₋₁₅acyl group thatmay be of straight chain, or where possible, of cyclic or branchedstructure and may contain one or more alkene, alkyne, or aromaticfunctionalities, and C₆H₄ is an aromatic ring, and —OR₁₆ is in a pararelationship to the (CH₂)_(m) group;

R⁸ and R⁹ in combination form a 5, 6 or 7 membered ring, which maycontain 1 or 2 heteroatoms selected from the group consisting of oxygen,nitrogen and sulfur, but otherwise contains only carbon and hydrogenatoms;

R¹⁰ is —(CH₂)_(m)—C₆₋₁₀aryl, or —(CH₂)_(m)—C₅₋₉heteroaryl wherein thearyl and heteroaryl may be monocyclic or bicyclic, a heteroaryl contains1 or 2 heteroatoms selected from oxygen, nitrogen and sulfur in the ringsystem, where R¹⁰ specifically includes the sidechain from phenylalanineand tryptophan;

R¹¹ is —(CH₂)_(n)—C₆₋₁₀aryl and —(CH₂)_(n)—C₅₋₉heteroaryl where aheteroaryl contains 1 or 2 heteroatoms selected from oxygen, nitrogenand sulfur in the ring system, and n is 1, 2, 3 or 4;

R¹² is —(CH₂)_(n)—C(═O)NH₂ wherein n is 1, 2, 3 or 4;

R¹³ is (CH₂)_(n)—C(═O)OR₁₇ wherein n is 1, 2, 3 or 4 and R₁₇ is ahydrogen atom; or a C₁₋₁₅alkyl group that may be of straight chain, orwhere possible, of cyclic or branched structure and may contain one ormore alkene, alkyne, or aromatic functionalities; or an aryl group; and

R¹⁴ is selected from the groups which may be R⁷ and R¹⁰ as describedabove, with the clarification that R¹4 need not be identically the sameas either R⁷ or R¹⁰.

The compounds of the present invention may be prepared in vitro, usingsolid phase or solution peptide synthesis techniques, or may be preparedin vivo, from microorganism ATCC 55797. Solution phase techniques as setforth in K. Okamato, K. et al. Bull. Chem. Soc. Jpn. 50:231-236 (1977),Ohno, M. et al. J. Am. Chem. Soc. 88(2):376-377 and Kosui, N. et al.Int. J. Peptide Protein Res. 18:127-134 (1981) may be modified toprepare the cyclic decapeptides of the present invention, merely byappropriate substitution of the suitably protected amino acids. Ösapay,G.; Profit, A.; Taylor, J. W., “Synthesis of Tyrocidine A. Use of OximeResin for Peptide Chain Assembly and Cyclization,” Tetrahedron Letters131(43):6121-6124 (1990) describes a synthetic scheme (termed PCOR forPeptide Cyclization on Oxine Resin) using a solid support, which can bemodified to prepare the compounds of the present invention. Thecompounds of the invention may also be isolated from microorganism ATCC55797 under appropriate conditions.

Ion exchange techniques can be used to prepare the various salts of theinvention, where such techniques are well known in the art. For example,hydrochloric acid may be added to a neutral compounds of the inventionto prepare the hydrochloride salt thereof. Dialysis techniques may alsobe employed to effect ion exchange and so obtain a desired salt of theinvention from another salt of the invention.

Derivatives of Loloatin A, B and C may be prepared simply by using thecorresponding derivatized amino acid in the synthesis of the cyclicdecapeptide. For example, if a Loloatin A derivative having tyrosinemethyl ether at the AA¹⁰ position is desired, such a cyclic decapeptidemay be prepared by the techniques described below, by substitutingtyrosine methyl ether for tyrosine. Other derivatives may be madeanalogously by techniques known in the art. Appropriate derivativedamino acid may be prepared by techniques known in the art, or they maybe purchased from any of several chemical supply houses, for example,Sigma Chemical and Bachem as identified elsewhere herein.

The Loloatins and compounds of the invention (i.e., the analogs andderivatives of Loloatins) have utility as antibiotics, and may be usedand administered in a manner analogous to antibiotics known in the art,to provide the beneficial effects desired of antibiotics. Preferably,the use is in the veterinary or, more preferably, the pharmaceuticalfield. Thus, the invention extends to the use of any compound of Formula(A or B) or Loloatin A, B or C for the manufacture of a medicament foruse in therapy. The invention further provides the use of any compoundof Formula (A or B) or Loloatin A, B or C for the mire of a medicamentfor use in the treatment of methicillin-resistant Staphylococcus aureus,vancomycin-resistant. Enterococcus faecalis, and Streptococcuspneumoniae infections in a mammal. The cyclic decapeptides of theinvention may be used against gram negative and gram positive bacteria.

In using a compound of Formula (A or B), or Loloatin A, B or C, thecompound is preferably administered to a patient in a pharmaceutical orveterinary composition comprising also a pharmaceutically orveterinarily acceptable carrier, and optionally, one or more otherbiologically active ingredients. Such compositions may be in any formused for oral, topical vaginal, parenteral, rectal and inhalatoryapplication. The compositions may be provided in discrete dose units.The carriers may be particulate, with compositions being, for example,tablets or powders, or liquid, with the compositions being, for example,oral syrups or injectable liquids, or gaseous, for inhalatoryapplication.

For oral administration, an excipient and/or binder may be present.Examples are sucrose, kaolin, glycerin, starch dextrins, sodiumalginate, carboxymethylcellulose and ethyl cellulose. Coloring and/orflavoring agents may be present. A coating shell may be employed. Forrectal administration oleaginous bases may be employed, for example,lanolin or cocoa butter. For an injectable formulation, buffers,stabilizers and isotonic agents may be included.

It will be evident to those of ordinary skill in the art that theoptimal dosage of the compounds of Formula (A or B), or Loloatin A, B orC may depend on the weight and physical condition of the patient; on theseverity and longevity of the illness; and on the particular form of theactive ingredient, the manner of administration and the compositionemployed.

It is to be understood that use of a compound of Formula (A or B), orLoloatin A, B or C in chemotherapy can involve such a compound beingbound to an agent, for example, a monoclonal or polyclonal antibody, aprotein or a liposome, which assist the delivery of said compound to thesite of infection.

Therefore, the invention relates further to a pharmaceutical orveterinary composition comprising an effective amount of compound ofFormula (A or B), or Loloatin A, B or C in association with a carrier.

In a further embodiment, the present invention provides a method for thetreatment of a patient afflicted with a bacterial infection comprisingthe administration thereto of a therapeutically effective amount of acompound of Formula (A or B), or Loloatin A, B or C.

The term “therapeutically effective amount” refers to an amount which iseffective, upon single or multiple dose administration to the patient,in providing relief of symptoms associated with bacterial infections. Asused herein, “relief of symptoms” of a bacterial infection refers to adecrease in severity over that expected in the absence of treatment anddoes not necessarily indicate a total elimination or cure of theinfection or condition caused thereby. In determining thetherapeutically effective amount or dose, a number of factors areconsidered by the attending diagnostician, including, but not limitedto: the species of mammal; its size, age, and general health; thespecific infection involved; the degree of or involvement or theseverity of the infection or condition arising therefrom; the responseof the individual patient; the particular compound administered; themode of administration; the bioavailability characteristics of thepreparation administered; the dose regimen selected; the use ofconcomitant medication; and other relevant circumstances.

A therapeutically effective amount of a compound of Formula (A or B), orLoloatin A, B or C is expected to vary from about 0.1 milligram perkilogram of body weight per day (mg/kg/day) to about 100 mg/kg/day.Preferred amounts are expected to vary from about 0.5 to about 10mg/kg/day.

In effecting treatment of a patient afflicted with a condition describedabove, a compound of Formula (A or B), or Loloatin A, B or C, can beadministered in any form or mode which makes the compound bioavailablein effective amounts, including oral, aerosol, and parenteral routes.For example, compounds of Formula (A or B), or Loloatin A, B or C, canbe administered orally, by aerosolization, subcutaneously,intramusculary, intravenously, transdermally, intranasally, rectally,topically, and the like. Oral or aerosol administration is generallypreferred. One skilled in the art of preparing formulations can readilyselect the proper form and mode of administration depending upon theparticular characteristics of the compound selected, the condition to betreated, the stage of the condition, and other relevant circumstances.See, e.g., Remington's Pharmaceutical Sciences, 18th Edition, MackPublishing Co. (1990).

The compounds can be administered alone or in the form of apharmaceutical composition in combination with pharmaceuticallyacceptable carriers or excipients, the proportion and nature of whichare determined by the solubility and chemical properties of the compoundselected, the chosen route of administration, and standardpharmaceutical practice.

In another embodiment, the present invention provides compositionscomprising a compound of Formula (A or B), or Loloatin A, B or C, inadmixture or otherwise in association with one or more inert carriers.These compositions are useful, for example, as assay standards, asconvenient means of making bulk shipments, or as pharmaceuticalcompositions. An assayable amount of a compound Formula (A or B), orLoloatin A, B or C, is an amount which is readily measurable by standardassay procedures and techniques as are well known and appreciated bythose skilled in the art. Assayable amounts of a compound Formula (A orB), or Loloatin A, B or C, will generally vary from about 0.001% toabout 75% of the composition by weight. Inert carriers can be anymaterial which does not degrade or otherwise covalently react with acompound of Formula (A or B), or Loloatin A, B or C,. Examples ofsuitable inert carriers are water, aqueous buffers, such as those whichare generally useful in High Performance Liquid Chromatography (HPLC)analysis, organic solvents, such as acetonitrile, ethyl acetate, hexaneand the like; and pharmaceutically acceptable carriers or excipients.

More particularly, the present invention provides pharmaceuticalcompositions comprising a therapeutically effective amount of a compoundof Formula (A or B), or Loloatin A, B or C, in admixture or otherwise inassociation with one or more pharmaceutically acceptable carriers orexcipients.

The pharmaceutical compositions are prepared in a manner well known inthe pharmaceutical art. The carrier or excipient may be a solid,semi-solid, or liquid material which can serve as a vehicle or mediumfor the active ingredient. Suitable carriers or excipients are wellknown in the art. The pharmaceutical composition may be adapted fororal, parenteral, or topical use and may be administered to the patientin the form of tablets, capsules, suppositories, solution, suspensions,or the like.

The compounds of the present invention may be administered orally, forexample, with an inert diluent or with an edible carrier. They may beenclosed in gelatin capsules or compressed into tablets. For the purposeof oral therapeutic administration, the compounds may be incorporatedwith excipients and used in the form of tablets, troches, capsules,elixirs, suspensions, syrups, wafers, chewing gums and the like. Thesepreparations should contain at least 4% of the compound of theinvention, the active ingredient, but may be varied depending upon theparticular form and may conveniently be between 4% to about 70% of theweight of the unit. The amount of the compound present in compositionsis such that a suitable dosage will be obtained. Preferred compositionsand preparations according to the present invention are prepared so thatan oral dosage unit form contains between 5.0-300 milligrams of acompound of the invention. The tablets, pills, capsules, troches and thelike may also contain one or more of the following adjuvants: binderssuch as microcrystalline cellulose, gum tragacanth or gelatin;excipients such as starch or lactose, disintegrating agents such asalginic acid, Primogel, corn starch and the like; lubricants such asmagnesium stearate or Sterotex; glidants such as colloidal silicondioxide; and sweetening agents such as sucrose or saccharin may be addedor a flavoring agent such as peppermint, methyl salicylate or orangeflavoring. When the dosage unit form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier such aspolyethylene glycol or a fatty oil. Other dosage unit forms may containother various materials which modify the physical form of the dosageunit, for example, as coatings. Thus, tablets or pills may be coatedwith sugar, shellac, or other enteric coating agents. A syrup maycontain, in addition to the present compounds, sucrose as a sweeteningagent and certain preservatives, dyes and colorings and flavors.Materials used in preparing these various compositions should bepharmaceutically pure and non-toxic in the amounts used.

For the purpose of parenteral therapeutic administration, the compoundsof the present invention may be incorporated into a solution orsuspension. These preparations should contain at least 0.1% of acompound of the invention, but may be varied to be between 0.1 and about50% of the weight thereof. The amount of the inventive compound presentin such compositions is such that a suitable dosage will be obtained.Preferred compositions and preparations according to the presentinvention are prepared so that a parenteral dosage unit contains between5.0 to 100 milligrams of the compound of the invention.

The compounds of Formula (A or B), or Loloatin A, B or C, of the presentinvention may also be administered by aerosol. The term aerosol is usedto denote a variety of systems ranging from those of colloidal nature tosystems consisting of pressurized packages. Delivery may be by aliquefied or compressed gas or by a suitable pump system which dispensesthe active ingredients. Aerosols of compounds of Formula (A or B), orLoloatin A, B or C, may be delivered in single phase, bi-phasic, ortri-phasic systems in order to deliver the active ingredient. Deliveryof the aerosol includes the necessary container, activators, valves,subcontainers, and the like. Preferred aerosols are able to bedetermined by one skilled in the art.

The compounds of Formula (A or B), or Loloatin A, B or C, of thisinvention may also be administered topically, and when done so thecarrier may suitably comprise a solution, ointment or gel base. Thebase, for example, may comprise one or more of the following:petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil diluentssuch as water and alcohol, and emulsifiers and stabilizers. Topicalformulations may contain a concentration of the Formula (A or B), orLoloatin A, B or C, compound of from about 0.1 to about 10% w/v (weightper unit volume).

The solutions or suspensions may also include one or more of thefollowing adjuvants: sterile diluents such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerin, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl paraben; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

The cyclic decapeptides of the invention may be combined with one ormore known antibiotics to provide a synergistic composition. In otherwords, a composition comprising a cyclic decapeptide of the inventionand a known antibiotic may have greater efficacy against bacteria thanwould be expected based on the individual efficacies of the cyclicdecapeptide and the known antibiotic.

As used herein, the term “patient” refers to a warm-blooded animal suchas a mammal which is afflicted with a particular inflammatory diseasestate. It is understood that guinea pigs, dogs, cats, rats, mice,horses, cattle, sheep, and humans are examples of animals within thescope of the meaning of the term.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

Symbols and abbreviations used herein are in accordance with therecommendation of IUPAC-IUB Commissioner on Biochemical Nomenclature, J.Biol. Chem. 1971, 247, 977. Abbreviations: “Asn” refers to asparagine;“Asp” refers to aspartic acid, “bp” refers to boiling point;BOP=benzotriazol-1-yl-oxy-tris-(dimethylamino) phosphoniumhexafluorophosphate; “BrZ” refers to bromobenzyloxycarbonyl; “Bzl”refers to benzyl; “° C” refers to degrees Celsius; CD=circulardichroism; “DCC” refers to N,N-dicyclohexylcarbodiimide;DCM=dichloromethane, DIEA=N,N-diisopropylethylamine;DMF=N,N-dimethylformamide; “DPPA” refers to diphenylphosphorylazide;FAB-MS=fast atom bombardment mass spectrometry; “HOBt” refers to1-hydroxybenzotriazole; “g” refers to grams; “Leu” refers to leucine;“mL” refers to milliliters; “mm Hg” refers to millimeters of mercury;“mmol” refers to millimoles; “NMP” refers to N-methylpyrrolidinone;“Orn” refers to ornithine; “Phe” refers to phenylalanine; “Pro” refersto proline; “TEA” refers to triethylaine; TMSOTf=trimethylsilyltrifiuoromethanesulfonate; “Tos” refers to p-toluenesulfonyl;. “Trp”refers to tryptophan; “Tyr” refers to tyrosine; “Val” refers to valine,Z=benzyloxycarbonyl; “μg” refers to micrograms; “μL” refers tomicroliters; and “μM” refers to micromolar.

A. PREPARATIVE EXAMPLES

The cyclic decapeptides of the present invention may be prepared invitro, using solid phase or solution peptide synthesis techniques, ormay be prepared in vivo, from microorganism ATCC 55797. Solution phasetechniques as set forth in K. Okamato, K. et al. Bull. Chem. Soc. Jpn.50:231-236 (1977), Ohno, M. et al. J. Am. Chem. Soc. 88(2):376-377 andKosui, N. et al. Int. J. Peptide Protein Res. 18:127-134 (1981) may bemodified to prepare the cyclic decapeptides of the present invention,merely by appropriate substitution of the suitably protected aminoacids.

The following examples present typical syntheses. These examples areunderstood to be illustrative only and are not intended to limit thescope of the present invention in any way.

Example A1

Ösapay, G.; Profit, A.; Taylor, J. W., “Synthesis of Tyrocidine A: Useof Oxime Resin for Peptide Chain Assembly and Cyclization,” TetrahedronLetters 131(43):6121-6124 (1990) describes a synthetic scheme (termedPCOR for Peptide Cyclization on Oxime Resin) using a solid support,which can be modified to prepare the cyclic decapeptides of the presentinvention. Further details of the PCOR method may be found in Ösapay,G.; Bouvier, M.; Taylor, J. W., “Peptide Cyclization on Oxime Resin(PCOR)” in Techniques in Protein Chemistry II, (ed. Villafranca, J. J.).The following description illustrates the synthesis of Loloatin A.

The p-nitrobenzophenone oxime polymer described by DeGrado and Kaisermay be used as a solid support in preparing cyclic decapeptides of thepresent invention. See DeGrado, W. F.; Kaiser, E. T., J. Org. Chem.45:1295-1300 (1980). For the preparation of for example,cyclo[L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-Tyr](Loloatin A) the starting compound Boc-Leu-resin is the same asdescribed in the Ösapay article, and the excess oxime groups may becapped by acetylation as described therein. The peptide chain may beassembled by consecutive addition of the following N^(α)-Boc-aminoacids, which have the L-configuration unless otherwise noted:BocOrn(Z)OH, BocValOK BocTyr(2,6-Cl₂-Bzl)OH, BocAsp(β-Bzl)OH, BocAsnOH,Boc-D-PheOH BocPheOH, BocProOH and BocTyr(2,6-Cl₂-Bzl)OH, all accordingto the BOP peptide coupling procedure of Fournier, A.; Wang, C. T.;Felix, A. M., Int. J. Pept., Prot. Res.; 31:86-97 (1988).

Boc protecting groups may be removed by treatment with 25% TFA/DCMsolution for 30 minutes. After the appropriate washing steps, Boc-aminoacids and BOP reagent may be added in 5-fold excess in DMF solutionfollowed by the same excess of DIEA. After a 2-hour reaction time, thecompleteness of each coupling may be monitored by the Kaiser test. SeeKaiser, E.; Colescott, R. L.; Cook, P. I., {dot over (A)}nal Biochem.,34:595-598 (1970). Coupling of BocAsnOH may be repeated with 2.5 molarequivalent reagent to insure a high yield of the product.

After final removal of the Boc protecting group from the N terminus, theamino group may be liberated from its TFA salt by addition of DIEA (1.5equivalents). The free amino group may cleave the peptide from thepolymer support by intrachain aminolysis in DCM at room temperature.After a 24 hour reaction time, the product may be obtained from thesolution phase by filtration. This crude product may be purified bysilica gel chromatography (e.g., 2×20 cm, eluentCHCl₃/MeOH/AcOH=18/1/1).

Protecting groups of the peptide may be removed with TMSOTf in TFA inthe presence of thioanisole, according to the procedure of Fujii, N. etal., J. Chem. Soc., Chem. Commun., 274-275 (1987). Hydrolysis of thepartly silylated product by NH₄OH may be followed by gel permeationchromatography, for example, using Sephadex G-10 column (eluent, e.g.,:2 M acetic acid in H₂O/MeOH, 4/1 [v/v]). Final purification may becarried out by RP-HPLC on, for example, a Vydac C₁₈ Proteinssemi-preparative column eluted at, e.g., 4 mL/min with a linear gradientof 25%-80% acetonitrile in 0.1% (v/v) TFA over 45 minutes.

In the above-described synthesis, one or more of the N^(α)-Boc-aminoacid starting materials may be purchased from chemical supply houses,for example, Sigma Chemical Company, PO Box 14508, St. Louis, Mo. 63178(Sigma's “Peptides and Amino Acids” catalog provides a convenientlisting) and Bachem, 6868 Nancy Ridge Dr., San Diego, Calif. 92121.

Example A2

The preparation of Loloatin B follows the synthesis of Loloatin A asdescribed in Example A1, with the exception that BocTyr(2,6-Cl₂-Bzl)OHis replaced with BocTrp(Z)OH or other sidechain amine-protectedL-tryptophan.

Example A3

The preparation of Loloatin C follows the synthesis of Loloatin B asdescribed in Example A2, with the exception that BocPheOH is replacedwith BocTrp(Z)OH or other sidechain amine-protected L-tryptophan.

Example A4

Solid phase peptide synthesis according to the method originallydescribed by Merrifield, J. Am Chem. Soc. 85:2149-2154, 1963, thedisclosure of which is hereby incorporated by reference, may be used toprepare the linear analogs of the cyclic decapeptides of the invention.Alternatively, solution synthesis may be used to prepare these linearpeptide analogs. Generally, peptides may be elongated by deprotectingthe α-amine of the C-terminal residue and coupling the next suitablyprotected amino acid through a peptide linkage using the methodsdescribed. This deprotection and coupling procedure is repeated untilthe desired sequence is obtained. This coupling can be performed withthe constituent amino acids in stepwise fashion, or by condensation offragments (two to several amino acids), or combination of bothprocesses, or by solid phase peptide synthesis as stated above.Thereafter, the linear peptides may be cyclized by well known peptidecycliation techniques, to prepare the cyclic decapeptides of theinvention.

When a solid phase synthetic approach is employed, the C-terminalcarboxylic acid is attached to an insoluble carrier (usuallypolystyrene). These insoluble carriers contain a group which will reactwith the α-carboxyl group to form a bond which is stable to theelongation conditions but readily cleaved later. Examples of whichinclude: chloro- or bromomethyl resin, hydroxymethyl resin, andaminomethyl resin. Many of these resins are commercially available withthe desired C-terminal amino acid already incorporated. Many of thesuitably protected amino acids used in the present invention are alsoavailable commercially from Sigma Chemical Company and Bachem.

Alternativey, compounds of the invention can be synthesized usingautomated peptide synthesizing equipment. In addition to the foregoing,peptide synthesis are described in Stewart and Young, “Solid PhasePeptide Synthesis,” 2nd ed., Pierce Chemical Co., Rockford, Ill. (1984);Gross Meienhofer, Udenfriend, Eds., “The Peptides: Analysis, Synthesis,Biology,” Vol. 1, 2, 3, 5 and 9, Academic Press, New York, 1980-1987;Bodanszky, “Peptide Chemistry: A Practical Textbook,” Springer-Verlag,N.Y. (1988); and Bodanszky et al., “The Practice of Peptide Synthesis,”Springer-Verlag, N.Y. (1984), the disclosures of which are herebyincorporated by reference.

Coupling between two amino acids, an amino acid and a peptide, or twopeptide fragments can be carried out using standard coupling proceduressuch as the azide method, mixed carbonic acid anhydride (isobutylchloroformate) method, carbodiimide (dicyclohexylcarbodiimide,diisopropylcarbodiimide, or water-soluble carbodiimide) method, activeester (p-nitrophenyl ester, N-hydroxy-succinic imido ester) method,Woodward reagent K method, carbonyldiimidazole method, phosphorusreagents such as BOP-Cl, or oxidation-reduction methods. Some of thesemethods (especially the carbodiimide method) can be enhanced by adding1-hydroxybenzotriazole. These coupling reactions can be performed ineither solution (liquid phase) or solid phase.

The functional groups of the constituent amino acids must be protectedduring the coupling reactions to avoid formation of undesired bonds. Theprotecting groups that can be used are listed in Greene, “ProtectiveGroups in Organic Chemistry,” John Wiley & Sons, New York (1981) and“The Peptides: Analysis, Synthesis, Biology,” Vol. 3, Academic Press,New York (1981), the disclosures of which are hereby incorporated byreference.

The α-carboxyl group of the C-terminal residue is usually protected byan ester that can be cleaved to give the carboxylic acid. Protectinggroups which can be used include: (1) alkyl esters such as methyl andt-butyl, (2) aryl esters such as benzyl and substituted benzyl, or (3)esters which can be cleaved by mild base treatment or mild reductivemeans such as trichloroethyl and phenacyl esters.

The α-amino group of each amino acid must be protected. Any protectinggroup known in the art can be used. Examples of which include: (1) acyltypes such as formyl, trifluoroacetyl, phthalyl, and p-toluenesulfonyl;(2) aromatic carbamate types such as benzyloxycarbonyl (Cbz or Z) andsubstituted benzyloxycarbonyls, 1-(p-biphenyl)1, -methylethoxy-carbonyl,and 9-fluorenylmethyloxycarbonyl (Fmoc); (3) aliphatic carbamate typessuch as tert-butyloxycarbonyl (Boc), ethoxycarbonyl,diisopropylmethoxycarbonyl, and allyloxycarbonyl; (4) cyclic alkylcarbamate types such as cyclopentyloxycarbonyl and adamantyloxycarbonyl;(5) alkyl types such as triphenylmethyl and benzyl; (6) trialkylsilanesuch as trimethylsilane; and (7) thiol containing types such asphenylthiocarbonyl and dithiasuccinoyl. The preferred α-amino protectinggroup is either Boc or Fmoc, preferably Fmoc. Many amino acidderivatives suitably protected for peptide synthesis are commerciallyavailable.

The α-amino protecting group is cleaved prior to the coupling of thenext amino acid. When the Boc group is used, the methods of choice aretrifluoroacetic acid, neat or in dichloromethane, or HCl in dioxane. Theresulting ammonium salt is then neutralized either prior to the couplingor in situ with basic solutions such as aqueous buffers, or tertiaryamines in dichloromethane or dimethylformamide. When the Fmoc group isused, the reagents of choice are piperidine or substituted piperidine indimethylformamide, but any secondary amine or aqueous basic solutionscan be used. The deprotection is carried out at a temperature between 0°C. and room temperature.

Any of the amino acid bearing side chain functionalities must beprotected during the preparation of the peptide using any of theabove-described groups. Those skilled in the art will appreciate thatthe selection and use of appropriate protecting groups for these sidechain functionalities depends upon the amino acid and presence of otherprotecting groups in the peptide. The selection of such protectinggroups is important in that it must not be removed during thedeprotection and coupling of the α-amino group. For example, when Boc isused as the α-amino protecting group, p-toluenesulfonyl (tosyl) moietiescan be used to protect the amino side chain of Orn. When Fmoc is chosenfor the α-amine protection usually tert-butyl based protecting groupsare acceptable. For instance, Boc can be used for ornithine.

Once the elongation of the peptide is completed all of the protectinggroups are removed. When a solution phase synthesis is used, theprotecting groups are removed in whatever manner is dictated by thechoice of protecting groups. These procedures are well known to thoseskilled in the art.

When a solid phase synthesis is used, the peptide is cleaved from theresin usually simultaneously with the protecting group removal. When theBoc protection scheme is used in the synthesis, treatment with anhydrousHF containing additives such as dimethyl sulfide, anisole, thioanisole,or p-cresol at 0° C. is the preferred method for cleaving the peptidefrom the resin. The cleavage of the peptide can also be accomplished byother acidic reagents such as trifluoromethanesulfonicacid/trifluoroacetic acid mixtures. If the Fmoc protection scheme isused, the N-terminal Fmoc group is cleaved with reagents describedearlier. The other protecting groups and the peptide are cleaved fromthe resin using a solution of trifluoroacetic acid and various additivessuch as anisole, etc.

Subsequent to removal of the linear peptide from the resin and removalof any protecting groups as desirable, the linear peptide is cyclizedusing conventional procedures such as by treatment with triethylamineand diphenylphosphorylazide in dimethylformamide. Prior to purificationof the crude cyclic peptide in the usual manner such as by use ofchromatography, any remaining protecting and functional group precursorsare removed or transformed into the desired group.

Example A4a Synthesis ofcyclo[L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-Lyr](Loloatin A) (SEQ ID NO:1).

Synthesis of the Linear Peptide

The linear analog of Loloatin A may be synthesized by standard solidphase methods using an Applied Biosystems (850 Lincoln Centre Dr.,Foster City, Calif.) 430A automated peptide synthesizer and protocolssupplied by the manufacturer. Commercially available Boc-amino acids maybe used with the following side chain protection: Orn(Tos), Tyr(BrZ),Asp(Bzl). Commercially available Boc-Val-PAM resin (0.5 mmol, AppliedBiosystems) may be deprotected with trifluoroacetic acid (2% anisole)and coupled in NMP with the HOBt esters of Boc-amino acids (4equivalents). The HOBt esters of Boc-amino acids may be formed by thereaction of the Boc-amino acid with DCC and HOBt. Couplings may becarried out for 30 minutes and the resin subsequently washed with NMPand DCM. Any unreacted amine may be acylated with acetic anhydride. Thedeprotection and coupling may be repeated until complete assembly of theprotected peptide resin is achieved. The linear peptide may besimultaneously deprotected and removed from the resin with anhydroushydrogen fluoride (10 mL) at 0° C. for 30 minutes in the presence ofanisole (5%). The peptide may then be extracted with 50% acetic acid,water and aqueous acetonitrile, and lyophilized.

Cyclization of the Linear Peptide

The crude linear peptide prepared above may be dissolved in DMF (˜5μmol/ml) and cyclized with DPPA (5 equivalents) TEA to adjust the pH to9. After completion of the reaction (4-48 hours), the solvent may beremoved and the crude cyclic peptide lyophilized fromacetonitrile/water. The cyclic peptide may then be desalted by gelfiltration in, e.g., 70% acetic acid over a Spectragel GF05 column(2.5×55 cm), and purified by reversed phase preparative HPLC (DynamaxC₁₈, 21.4×250 mm, Rainin) using various gradients of 0.1% aqueous TFAand acetonitrile.

The purified peptide may then be characterized by Analytical HPLC (Vydac218TP54, 4.6×250 mm), FAB-MS and amino acid analysis, and thesecharacterizations compared to theoretical values when available.

Example A5

The procedure of Example A4a may be modified to prepare Loloatin B.Thus, when Tyr(BrZ) is replaced with Trp(Z) or other amine-protectedL-tryptophan, Loloatin B may be prepared.

Example A6

The procedure of Example A5 may be modified to prepare Loloatin C. Thus,when Phe is replaced with Trp(Z) or other amine-protected L-tryptophan,Loloatin C may be prepared.

Example A7

Each of Loloatin A, B and C may be isolated after fermentation of ATCC55797 as described below.

The marine bacterial isolate MK-PNG-276A, tentatively identified as aBacillus laterosporus by MIDI analysis of cellular fatty acids, wasobtained from the tissues of an unidentified tube worm collected at −15m off of Loloata Island, Papua, New Guinea. MK-PNG-276A has beendeposited with the American Type Culture Collection as ATCC 55797.

MK-PNG-276A was cultured on trays of solid tryptic soy agar supplementalwith NaCl to a concentration of 1%. Twenty-six 400 mL trays (9″×15″×¼″deep agar) were cultured for five days after which the combined cellsand agar were lyophilized. The lyophilization product, (61.5 g dryweight) was extracted with three 600 mL portions of methanol that werecombined, filtered, and reduced in vacuo to give a brown/gray tar. Thetar was dissolved in 750 mL of MeOH/H₂O (¼) and sequentially extractedwith hexanes (3×250 mL) and EtOAc (3×250 mL). The combined EtOAcextracts were reduced in vacuo to give a taupe/brown crystalline solid(5.5 g). The EtOAc residue was then processed in batches. Sizeseparation on an LH-20 Sephadex column with methanol eluant gave sixfractions. The first and major fraction showed antibiotic activityagainst MRSA and Enterococci species. This fraction was then subjectedto preparative reverse phrase column chromatography and RP HPLC using9:1 methanol/water containing 0.1% TFA, to yield Loloatin A (relativeretention time on HPLC=0.70, white powder 281 mg, 0.45% dry wt ofcells), Loloatin B (relative retention time on HPLC=1.00, tan/whitepowder solid 1.87 g, 3.0% dry wt of cells) and Loloatin C (relativeretention time on HPLC=0.66, tan/white powder 40 mg, 0.065% dry wt ofcells).

Loloatin B (2) gave a (M+H) ion in the HRFABMS at m/z 1296.64232appropriate for a molecular formula of C₆₇H₈₅N₁₃O₁₄ (ΔM+0.46 ppm).Detailed analysis of the ¹H, ¹³C, COSY, HOHAHA, HMQC, HMBC and ROESYdata for Loloatin B (2) identified the ten amino acids residuesindicated in Table 1. Hydrolysis of 2 at 100° C. with 6N HCl containingthioglycolic acid and examination of the pentafluropropionamideisopropyl ester derivatives of the liberated amino acids via chiral GCanalysis confirmed the presence of L-valine, L-ornithine, L-leucine,D-tyrosine, L-proline, L-phenylalanine, D-phenylalanine, L-tryptophanand L-aspartic acid (from ASP and ASN). The ten identified amino acidresidues accounted for all of the atoms in the molecular formula of (2),and 31 of the 32 sites of unsaturation demanded by the molecularformula. Thus, Loloatin B (2) had to be a monocyclic decapeptide.

The amino acid sequence in (2) was determined by analysis of HMBC andROESY data HRFABMS and MS/MS studies supported the amino acid sequencederived from the NMR data. The MS/MS data was consistent with initialcleavage of the ring at the TYR-CO/PRO-N bond to give a lineardecapeptide LEU-TYR (m/z 1019), ORN-LEU-TYR (m/z 905) andTRP-VAL-ORN-TYR (SEQ ID NO:78) (m/z 621). FABMS peaks at m/z 245 and 377could be assigned to the protonated fragments PRO-PHE1 and PHE2-ASN-ASP,respectively.

The amino acid sequences for Loloatin A and C were determined in ananalogous manner.

TABLE 1 ¹H NMR Data (500 MHz) for Loloatin B (2) (DMSO-D₆) Res. δ ^(1H)VAL NH 7.52(d, J=8.0) αCH 4.56(m) βCH 2.01(sept, J=7.0) γCH₃ 0.93(d)γCH₃ 0.93(d) CO — ORN NH 8.88(d,J=8.9) αCH 5.27(bm) βCH₂ 1.8(m) γCH₂1.7(m) δCH₂ 2.8, 2.9(m) δNH 7.45(bs) CO — CH₃ CO LEU NH 7.92(bs) αCH4.55(m) βCH₂ 1.35(m) 1.25(m) γCH 1.5(m) δCH₃ 0.93(d) δCH₃ 0.93(d) CO —TYR NH 9.21(s) αCH 4.22(m) βCH₂ 2.70(m) 2.81(m) iC — oCH 6.98(d, J=8.6)mCH 6.61(d, J=8.6) pCOH not observed CO — PRO N — αCH 4.07(d, J=7.6)βCH₂ 1.43(m) 1.25(m) γCH₂ 1.07(m) 0.411(m) δCH₂ 2.20(m) 3.30(m) CO —PHE1 NH 7.23(d, J=9.5) αCH 4.5(m) PHE1 CH₂ 2.25(m) iC — oCH 7.18 mCH pCHCO — PHE2 NH 9.05(d, J=9.0) αCH 5.57(bt) βCH₂ 2.75(m) 3.02(m) iC — oCH7.18 mCH pCH CO — ASN NH 9.03(d, J=6.0) αCH 4.49(m) βCH₂ 3.37(m) 3.0(m)CO — NH₂ 8.04(bs) 7.45 CO — ASP NH 8.32(d, J=4.2) αCH 4.26(m) βCH₂2.35(m) 2.25(m) γCO — CH₃ CO — TRP NH 8.63(d, J=9.8) αCH 4.5(m) βCH₂3.15(m) C — C — CH 7.5 CH 7.0 CH 7.03(m) CH 7.32(J=8.0) C — NH 10.81(bs)CH 7.02(d) CO —

Example A8

Fermentation techniques with the microoganism ATCC 55797 may be used toobtain the cyclic decapeptides of Formulas 1, 2 and 3 as described inExample A7. Thereafter, the cyclic decapeptides of Formulas 1, 2 and 3may be derivatized to form salts (either acid- or base- addition salts,depending on whether the amino acid sidechain is basic or acidic,respectively), esters (from amino acid sidechains containing acarboxylic acid group), amines (from amino acid sidechains containing anamino group), ethers (from amino acid sidechains containing an hydroxylgroup) and amides (from amino acid sidechains containing either an amineor carboxylic acid group) of the invention.

N-acetyl loloatin B methyl ester (276bs2): Loloatin B (276bs1) (100 mg)was acetylated under argon, at 23° C. for 16 hours, using 2 mL of aceticanhydride and 1 mL anhydrous pyridine (freshly distilled). The solutionwas reduced in vacuo and the crude acetylated material was then loadedonto a reverse phase ODS sep-pak™ using 5 mL of 1:1 methanol/water,followed by elution with 5 mL of methanol. The methanol eluant wasreduced in vacuo, dissolved in 5 mL tetrahydrofuran and reacted withdiazomethane in a micro molar generator using a dry ice/acetone bath tocool the THF solution. N-acetyl loloatin B methyl ester (276bs2) waspurified using reverse phase ODS HPLC with 17:3 methanol/water as eluantto yield 30 mg of N-acetyl loloatin B methyl ester.

Melting point: 229-233° C.; IR (Nujol mull) υ_(max): 3278 (br,m), 3032(m), 3065 (w), 1636 (s), 1537 (s), 1251 br,m); [a]_(D)-62.7 (ETOH); UV(EtOH) λ_(max) (e): 276 (13,000), 228 (45,800), 222 (52,600), 218(52,900), 214 (47,500), 208 (36,100); HRFABMS (M+H) m/z 1296.64232(C₆₇H₈₆N₁₃O₁₄; ΔM 0.46 ppm). Chiral GC analysis of a 6N HCl hydrolysateof loloatin B identified L-leucine, L-proline, L-phenylalanine,D-phenylaline, L-ornithine, L-valine, L-tryptophan, D-tyrosine,L-aspartic acid, and L-asparagine.

Loloatin B (2) gave a (M+H) ion in the HRFABMS at m/z 1296.64232appropriate for a molecular formula of C₆₇H₈₅N₁₃O₁₄ (ΔM+0.46 ppm).Detailed analysis of the ¹H, ¹³C, COSY, HOHAHA, HMQC, HMBC and ROESYdata for loloatin B (2) and its N-acetyl methyl ester derivative (7),identified the ten amino acids residues indicated in Table 1. Hydrolysisof 2 at 100° C. with 6N HCl containing thioglycolic acid and examinationof the pentafluropropionamide isopropyl ester derivatives of theliberated amino acids via chiral GC analysis confirmed the presence ofL-valine, L-ornithine, Leucine, D-tyrosine, L-proline, L-phenylalanine,D-phenylalanine, L-tryptophan and L-aspartic acid (from ASP and ASN).The ten identified amino acid residues accounted for all of the atoms inthe molecular formula of (2), and 31 of the 32 sites of unsaturationdemanded by the molecular formula. Thus, loloatin B (2) had to be amonocyclic decapeptide.

The amino acid sequence in (2) was determined by analysis of the HMBCand ROESY data for both (2) and (7), as shown in FIG. 1. ROESYcorrelations observed in (7) between amino acid α-methine protons andadjacent residue NH protons unambiguously identified the following fiveamino bonds: ORN-CO/LEU-NH (δ 5.36/8.22), ASP-CO/TRP-NH (δ 4.40/8.72),ASN-CO/ASP-NH (δ 4.55/8.36), PHE2-CO/ASN-NH (δ 5.73/9.13) andPRO-CO/PHE1-NH (δ 4.18/7.39). A ROESY correlation observed between theTYR (α-methine resonance at δ 4.25 and the PRO δ-proton resonance at δ3.30 in 7 identified the TYR-CO/PRO-N amide bond, and a strong ROESYcorrelation between the VAL-NH resonance at δ 7.52 and the TRP-NHresonance at δ 8.63 in 2 identified the TRP-CO/VAL-NH amide linkage. Thelatter ROESY correlation suggests the possibility of a β-bulge in theTRP/VAL region of the cyclic peptide as shown in FIG. 1. See, e.g., Kuo,M. et al., J. Am. Chem. Soc. 102:520-24, 1980; Eggleston, D. S. et al.,J. Am. Chem. Soc. 113:4410, 1991; and Peishoff, C. E. et al., J. Am.Chem. Soc. 113:4416, 1991. HMBC correlations from both the VAL β-methineproton resonance at δ 2.12 and the ORN-NH resonance at δ 8.81 to thewell resolved carbonyl resonance at δ 169.6 in derivative 7 identifiedthe VAL-CO/ORN-NH amide bond, and HMBC correlations from both the LEU δmethylene proton resonance at δ 1.25 and the TYR-NH resonance at δ 9.21to the carbonyl resonance at δ 171.8 in 2 identified the LEU-CO/T-NHamide bond. The final PHE1-CO/PHE2-NH amide bond was required tocomplete the macrocyclic ring.

BRFABMS and MS/MS studies supported the amino acid sequence derived fromthe NMR data. The MS/MS data was consistent with initial cleavage of thering at the TYR-CO/PRO-N bond to give a linear decapeptide thatsequentially loses LEU-TYR (m/z 1019), ORN-LEU-TYR (m/z 905) andTRP-VAL-ORN-LEU-TYR (m/z 621). FABMS peaks at m/z 245 and 377 could beassigned to the protonated fragments PRO-PHE1 and PHE2-ASN-ASP,respectively.

Example A9

Precursor directed biosynthesis, wherein a culture media containing themicroorganism isolated in Example A7 is supplemented with a replacementamino acid at fairly high concentrations, may be used to prepare cyclicdecapeptides of the invention. See, e.g., Katz, E. and Demain, A. L.,“The Peptide Antibiotics of Bacillus: Chemistry Biogenesis, and PossibleFunctions,” Bacteriological Reviews, June 1977, pp. 449-474.

The culture media described in Example A7 can be employed in precursordirected biosynithesis to prepare the analogs of Loloatin A, B and C byproviding to the microorganism ATCC 55797 fairly high concentrations ofthe following amino acids. Butyrine may replace valine. L-diaminobutyricacid may replace ornithine. Any of L-isoleucine, L-alloisoleucine,L-norvaline, L-cyclopropylalanine and norleucine may replace leucine.Any of p-fluorophenyalanine, tryptophan and thienylalanine may replacetyrosine. Any of azetidine-2-carboxylic acid, pipecolic acid,trans-3-methylproline and trans-4-fluoroproline may replace proline. Anyof tyrosine, p-fluorophenyalanine, tryptophan, thienylalanine andβ-phenylserine may replace phenylalanine. Any of tyrosine,p-fluorophenyalanine, phenylalanine, thienylalanine and β-phenylserinemay replace tryptophan.

Example A10 Synthesis of Loloatin B

Linear precursors were prepared, and then those precursors were cyclizedto provide Loloatin B. The structure of the linear precursor is selectedin order that the geometry of the linear sequence will bring togetherthe N- and C- termini, thus favoring the cyclization reaction. Thelinear precursor was prepared by standard solid phase peptide synthesis(i.e., typical coupling and cleavage conditions), using a standardpeptide acid resin (Wang), to provide:

NH₂ OL dY PF dF NDWV-COOH

Several cyclization chemistries were explored with this fillydeprotected linear peptide precursor.

A. NHS-Osu yields very slow reaction EDC multiple products B. HOBT/EDCmultiple, very hydrophobic 10 equiv, very dilute products (oligomers) C.HOBT 4 equiv 0.5 mg/ml, 20% DMF/methylene chloride EDC 5 equiv 4 hourlyadditions of equal aliquots. Cleanest of the conditions.

The complex reaction mixture from Method C was chromatographed onreverse phase HPLC, and a microtiter plate assay was run throughout theentire chromatogram to identify active materials. Only one fraction, fr73, (1 mg yield of starting 100 mg) exhibited the desired biologicalactivity.

In-vitro Microbiology MRSA 034 P.a. 028 MIC MBZ MIC MBZ Fraction 73 8.08.0 >128 >128

Fraction 73 was identified as Loloatin B by mass spectroscopy and NMRspectroscopy. Fraction 73 did not have a free N-terminal amino group.

Loloatin B was also prepared from the following precursor, which wasprepared by standard solid-phase synthesis techniques:

dF NDWVOL dY PF-COOH

The cyclization of this compound was carried out as in Method C. Withthis compound, cyclization proceeded much more slowly and yieldedproportionately more lower r.t. materials than the first cyclization andless of the materials in the desired portion of the chromatogram.

B. ACTIVITY EXAMPLES Example B1

In a standard liquid dilution antimicrobial assay described below,Loloatin B was found to be selectively antimcrobial with the minimuminhibitory concentrations listed in Table 2 below. Antimicrobialactivity was determined by macrobroth dilution antimicrobialsusceptibility testing. A solution of Loloatin B was prepared in trypticsoy broth. Initially, a 100 μmg/ml solution of the peptide was tested.If inhibition of a target microorganism was detected, serial two-folddilutions (in broth) of the Loloatin B solutions were tested todetermine the minimal inhibitory concentration (MIC) of Loloatin B foreach target organism Target organisms tested are identified in Table 2.Turbidity standardized suspensions of each organism were preparedaccording to accepted protocols using a 0.5 McFarland turbiditystandard, and these standardized suspensions were used to inoculate atube containing Loloatin B. Activity of Loloatin B was indicated by lackof growth (turbidity) of one or more of the target organisms.

See Woods, G. L. et al., “Antibacterial susceptibility tests: dilutionand disk diffusion methods, Manual of Clinical Microbiology (6^(th)Ed.),Murray, Baron, Pfaller, Tenover and Yolken (Eds.), ASM Press, WashingtonD.C., 1995, pp. 1327-1341.

TABLE 2 Minimum Inhibitory Concentrations of Loloatin B Against a Panelof Human Pathogens Methicillin resistant Staphylococcus aureus  <2 μg/mLVancomycin resistant Enterococcus faecalis  <2 μg/mL Penicillinresistant Streptococcus pneumoniae  <2 μg/mL Candida albicans >100 μg/mLPseudomonas aeruginosa >100 μg/mL Enterobacter cloacae >100 μg/mLXanthomonas maltophilia >100 μg/mL Escherichia coli >100 μg/mL

Example B2

Antimicrobial activity was determined by macrobroth dilutionantimicrobial susceptibility testing. Solutions of the cyclicdecapeptides (Loloatin A, B and C) and a control (vancomycin) wereprepared in tryptic soy broth. Initially, a 100 μg/ml solution of eachcompound was tested. When inhibition of target microorganism wasdetected, serial two-fold dilutions of the compound in broth were testedto determine the minimal inhibitory concentration (MIC) of each compoundfor each target organisms. Target organisms tested are listed below inTable 3, and included methicillin resistant Staphylococcus aureus,vancomycin resistant Enterococcus sp. Escherichia coli, multiply drugresistant Pseudomonas aeruginosa; Stenofrophomonas maltophilia andCandida albicans. Turbidity standardized suspensions of each organismwere prepared according to accepted protocols using a 0.5 McFarlandturbidity standard, and these standardized suspensions were used toinoculate the tubes containing the compounds. Activity of a compound wasindicated by lack of growth (turbidity) of one or more of the targetorganisms. See Woods, G. L. and Washington, J. A., “Antibacterialsusceptibility tests: dilution and disk diffusion methods, Manual ofClinical Microbiology (6^(th) Ed.), Murray, Baron, Pfaller, Tenover andYolken Eds.), ASM Press, Washington D.C., 1995, pp. 1327-1341. Othercompounds of the invention may be screened and evaluated for antibioticactivity in the same manner.

The data of Table 3 demonstrates that Loloatin C has excellent activityagainst Escherichia coli, a gram negative rod bacteria.

TABLE 3 ANTIBACTERIAL ACTIVITY OF THE LOLOATINS (BROTH DILUTION MICS INμG/ML) Loloatin Loloatin Loloatin Vanco- A B C mycin S. aureus (MRSA)2-4 2-4 0.5-1   Enterococcus faecalis 2-4 2-4 2-4 20 (ATCC51299)Enterococcus faecalis (13242) 2-4 2-4 1-2 >100 Enterococcus faecium(F4641) 2-4 2-4 2 6 Enterococcus faecium (19007) 2-4 2-4 2 >100Enterococcus gallinarum 2-4 2-4 1-2 6 Group A Streptococcus (19615)<0.25 <0.25 <0.25 Group B Streptococcus (12401) <1 <1 <0.25 Candidaalbicans 16 16 16 Pseudomonas aeruginosa >100 #100 >100 Enterobactercloacae >100 >100 >100 Stenotrophomonas maltophilia >100 >100 >100Escherichia coli >100 >100 1-2

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually incorporated by reference.

From the foregoing, it will be evident that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

78 1 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 1 Val Xaa LeuTyr Pro Phe Phe Asn Asp Tyr 1 5 10 2 10 PRT Bacillus laterosporusMOD_RES (2)...(2) Orn 2 Val Xaa Leu Tyr Pro Phe Phe Asn Asp Trp 1 5 10 310 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 3 Val Xaa Leu Tyr ProTrp Phe Asn Asp Trp 1 5 10 4 10 PRT Bacillus laterosporus MOD_RES(1)...(1) Butyrine 4 Xaa Xaa Leu Tyr Pro Phe Phe Asn Asp Tyr 1 5 10 5 10PRT Bacillus laterosporus MOD_RES (1)...(1) Butyrine 5 Xaa Xaa Leu TyrPro Phe Phe Asn Asp Trp 1 5 10 6 10 PRT Bacillus laterosporus MOD_RES(1)...(1) Butyrine 6 Xaa Xaa Leu Tyr Pro Trp Phe Asn Asp Trp 1 5 10 7 10PRT Bacillus laterosporus MOD_RES (2)...(2) Dbu 7 Val Xaa Leu Tyr ProPhe Phe Asn Asp Tyr 1 5 10 8 10 PRT Bacillus laterosporus MOD_RES(2)...(2) Dbu 8 Val Xaa Leu Tyr Pro Phe Phe Asn Asp Trp 1 5 10 9 10 PRTBacillus laterosporus MOD_RES (2)...(2) Dbu 9 Val Xaa Leu Tyr Pro TrpPhe Asn Asp Trp 1 5 10 10 10 PRT Bacillus laterosporus MOD_RES (2)...(2)Orn 10 Val Xaa Ile Tyr Pro Phe Phe Asn Asp Tyr 1 5 10 11 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 11 Val Xaa Ile Tyr Pro Phe Phe AsnAsp Trp 1 5 10 12 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 12Val Xaa Ile Tyr Pro Trp Phe Asn Asp Trp 1 5 10 13 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 13 Val Xaa Xaa Tyr Pro Phe Phe AsnAsp Tyr 1 5 10 14 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 14Val Xaa Xaa Tyr Pro Phe Phe Asn Asp Trp 1 5 10 15 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 15 Val Xaa Xaa Tyr Pro Trp Phe AsnAsp Trp 1 5 10 16 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 16Val Xaa Xaa Tyr Pro Phe Phe Asn Asp Tyr 1 5 10 17 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 17 Val Xaa Xaa Tyr Pro Phe Phe AsnAsp Trp 1 5 10 18 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 18Val Xaa Xaa Tyr Pro Trp Phe Asn Asp Trp 1 5 10 19 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 19 Val Xaa Xaa Tyr Pro Phe Phe AsnAsp Tyr 1 5 10 20 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 20Val Xaa Xaa Tyr Pro Phe Phe Asn Asp Trp 1 5 10 21 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 21 Val Xaa Xaa Tyr Pro Trp Phe AsnAsp Trp 1 5 10 22 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 22Val Xaa Xaa Tyr Pro Phe Phe Asn Asp Tyr 1 5 10 23 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 23 Val Xaa Xaa Tyr Pro Phe Phe AsnAsp Trp 1 5 10 24 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 24Val Xaa Xaa Tyr Pro Trp Phe Asn Asp Trp 1 5 10 25 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 25 Val Xaa Leu Xaa Pro Phe Phe AsnAsp Tyr 1 5 10 26 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 26Val Xaa Leu Xaa Pro Phe Phe Asn Asp Trp 1 5 10 27 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 27 Val Xaa Leu Xaa Pro Trp Phe AsnAsp Trp 1 5 10 28 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 28Val Xaa Leu Trp Pro Phe Phe Asn Asp Tyr 1 5 10 29 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 29 Val Xaa Leu Trp Pro Phe Phe AsnAsp Trp 1 5 10 30 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 30Val Xaa Leu Trp Pro Trp Phe Asn Asp Trp 1 5 10 31 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 31 Val Xaa Leu Xaa Pro Phe Phe AsnAsp Tyr 1 5 10 32 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 32Val Xaa Leu Xaa Pro Phe Phe Asn Asp Trp 1 5 10 33 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 33 Val Xaa Leu Xaa Pro Trp Phe AsnAsp Trp 1 5 10 34 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 34Val Xaa Leu Tyr Xaa Phe Phe Asn Asp Tyr 1 5 10 35 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 35 Val Xaa Leu Tyr Xaa Phe Phe AsnAsp Trp 1 5 10 36 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 36Val Xaa Leu Tyr Xaa Trp Phe Asn Asp Trp 1 5 10 37 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 37 Val Xaa Leu Tyr Xaa Phe Phe AsnAsp Tyr 1 5 10 38 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 38Val Xaa Leu Tyr Xaa Phe Phe Asn Asp Trp 1 5 10 39 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 39 Val Xaa Leu Tyr Xaa Trp Phe AsnAsp Trp 1 5 10 40 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 40Val Xaa Leu Tyr Xaa Phe Phe Asn Asp Tyr 1 5 10 41 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 41 Val Xaa Leu Tyr Xaa Phe Phe AsnAsp Trp 1 5 10 42 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 42Val Xaa Leu Tyr Xaa Trp Phe Asn Asp Trp 1 5 10 43 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 43 Val Xaa Leu Tyr Xaa Phe Phe AsnAsp Tyr 1 5 10 44 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 44Val Xaa Leu Tyr Xaa Phe Phe Asn Asp Trp 1 5 10 45 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 45 Val Xaa Leu Tyr Xaa Trp Phe AsnAsp Trp 1 5 10 46 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 46Val Xaa Leu Tyr Pro Tyr Phe Asn Asp Tyr 1 5 10 47 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 47 Val Xaa Leu Tyr Pro Tyr Phe AsnAsp Trp 1 5 10 48 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 48Val Xaa Leu Tyr Pro Xaa Phe Asn Asp Tyr 1 5 10 49 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 49 Val Xaa Leu Tyr Pro Xaa Phe AsnAsp Trp 1 5 10 50 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 50Val Xaa Leu Tyr Pro Trp Phe Asn Asp Tyr 1 5 10 51 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 51 Val Xaa Leu Tyr Pro Trp Phe AsnAsp Trp 1 5 10 52 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 52Val Xaa Lys Tyr Pro Xaa Phe Asn Asp Tyr 1 5 10 53 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 53 Val Xaa Leu Tyr Pro Xaa Phe AsnAsp Trp 1 5 10 54 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 54Val Xaa Leu Tyr Pro Xaa Phe Asn Asp Tyr 1 5 10 55 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 55 Val Xaa Leu Tyr Pro Xaa Phe AsnAsp Trp 1 5 10 56 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 56Val Xaa Leu Tyr Pro Phe Tyr Asn Asp Tyr 1 5 10 57 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 57 Val Xaa Leu Tyr Pro Phe Tyr AsnAsp Trp 1 5 10 58 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 58Val Xaa Leu Tyr Pro Phe Xaa Asn Asp Tyr 1 5 10 59 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 59 Val Xaa Leu Tyr Pro Phe Xaa AsnAsp Trp 1 5 10 60 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 60Val Xaa Leu Tyr Pro Phe Trp Asn Asp Tyr 1 5 10 61 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 61 Val Xaa Leu Tyr Pro Phe Trp AsnAsp Trp 1 5 10 62 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 62Val Xaa Leu Tyr Pro Phe Xaa Asn Asp Tyr 1 5 10 63 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 63 Val Xaa Leu Tyr Pro Phe Xaa AsnAsp Trp 1 5 10 64 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 64Val Xaa Leu Tyr Pro Phe Xaa Asn Asp Tyr 1 5 10 65 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 65 Val Xaa Leu Tyr Pro Phe Xaa AsnAsp Trp 1 5 10 66 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 66Val Xaa Leu Tyr Pro Trp Phe Asn Asp Tyr 1 5 10 67 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 67 Val Xaa Leu Tyr Pro Phe Phe AsnAsp Xaa 1 5 10 68 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 68Val Xaa Leu Tyr Pro Trp Phe Asn Asp Xaa 1 5 10 69 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 69 Val Xaa Leu Tyr Pro Phe Phe AsnAsp Phe 1 5 10 70 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 70Val Xaa Leu Tyr Pro Trp Phe Asn Asp Phe 1 5 10 71 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 71 Val Xaa Leu Tyr Pro Phe Phe AsnAsp Xaa 1 5 10 72 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 72Val Xaa Leu Tyr Pro Trp Phe Asn Asp Xaa 1 5 10 73 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 73 Val Xaa Leu Tyr Pro Phe Phe AsnAsp Xaa 1 5 10 74 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 74Val Xaa Leu Tyr Pro Trp Phe Asn Asp Xaa 1 5 10 75 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 75 Val Xaa Leu Tyr Pro Phe Phe AsnAsp Tyr 1 5 10 76 10 PRT Bacillus laterosporus MOD_RES (2)...(2) Orn 76Val Xaa Leu Tyr Pro Phe Phe Asn Asp Trp 1 5 10 77 10 PRT Bacilluslaterosporus MOD_RES (2)...(2) Orn 77 Val Xaa Leu Tyr Pro Trp Phe AsnAsp Trp 1 5 10 78 5 PRT Bacillus laterosporus MOD_RES (3)...(3) Orn 78Trp Val Xaa Leu Tyr 1 5

What is claimed is:
 1. An isolated compound selected from the groupconsisting of: cyclo[L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-Tyr] (SEQ IDNO:1); cyclo[L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-Trp] (SEQ IDNO:2); and cyclo[L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Trp-D-Phe-L-Asn-L-Asp-L-Trp] (SEQ IDNO:3).
 2. A compound of claim 1 having the formula: cyclo[L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-Tyr] (SEQ IDNO:1).
 3. A compound of claim 1 having the formula: cyclo[L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-Trp] (SEQ IDNO:2).
 4. A compound of claim 1 having the formula: cyclo[L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Trp-D-Phe-L-Asn-L-Asp-L-Trp] (SEQ IDNO:3).
 5. A pharmaceutical composition comprising a compound of any ofclaims 1, 2, 3, or 4, and a pharmaceutically acceptable carrier.
 6. Amethod for the treatment of a patient afflicted with a bacterialinfection comprising the administration to said patient of atherapeutically effective amount of a compound of any of claims 1, 2, 3,or 4.